How mixing honey bee drone semen affects sperm quality: a preliminary study
Preface
After mating, sperm from multiple Apis mellifera drones mix in the queen’s spermatheca. Their behaviour there matters for long-term sperm survival and successful fertilisation. The honey bee’s polyandrous mating system may also allow cryptic female choice and sperm competition to favour traits that contribute to successful reproduction. Cryptic female choice refers to mechanisms by which a female can influence the use of sperm from different males after mating, for example by retaining or expelling different proportions of their ejaculates.
This preliminary study measured several sperm quality traits in mixed drone semen. The researchers collected 31 ejaculates and split them to prepare samples with semen from one, two or three drones. They used computer-assisted sperm analysis at baseline and after 60 minutes. Total motility declined significantly in two-drone samples (p = 0.004). Three-drone samples tended to show lower motility and kinematic values at baseline, without a significant difference between groups in those baseline measures. After an hour, the three-drone group alone showed increases in progressive motility, swimming speed and kinematic measures. Sperm concentration correlated with most functional measures, whereas morphometric measurements did not. The observed changes may reflect the combined effects of mixing, sperm concentration and time available for sperm interactions.
Introduction
In polyandrous insects, a female mates with several males and receives semen from each. Among Hymenoptera, this can increase genetic diversity and benefit colony health and fitness. The honey bee (Apis mellifera) is an example of extreme polyandry. In some populations, a queen may mate with as many as 77 drones (Withrow and Tarpy 2018; Delaplane et al. 2021). The benefits associated with polyandry include improved worker performance and productivity, more efficient foraging, greater brood survival and larger colonies (Mattila and Seeley 2007; Delaplane et al. 2021). Multiple mating can also reduce the risk that a queen exhausts her sperm reserves (Delaplane et al. 2021). It may help provide enough spermatozoa with traits that support long-term fertilisation. The Cape honey bee, A. m. capensis, has smaller drones with lower sperm counts and queens with a larger spermatheca than European subspecies such as A. m. carnica. This may increase the number of matings needed for a lifetime supply of sperm (Kraus et al. 2004).
Receiving large numbers of spermatozoa from multiple males may help ensure an adequate supply of viable sperm. Unlike queens, honey bee drones mate only once. After mating, sperm survival depends on conditions in the queen (den Boer et al. 2010). About 4–6 million spermatozoa reach and remain in the spermatheca, roughly 3–5% of the total transferred by the drones and 5–10% of an individual drone’s contribution according to the cited estimates. The remainder is expelled through the bursa copulatrix (Baer 2005; Gençer and Kahya 2020; Liu et al. 2020; Gençer et al. 2014). An estimated 1–1.6 million spermatozoa are eventually used to fertilise eggs over the following years (Baer 2005). Their ability to remain viable over that time makes sperm quality important (Baer 2003; Stürup et al. 2013). Researchers have therefore explored sperm competition in honey bees (Moritz 1986; Woyciechowski and Król 1996; Shafir et al. 2009). Competition among males and cryptic female choice can shape reproductive outcomes before or after mating.
After mating, sperm competition has been proposed in both the oviducts and the spermatheca (Gençer and Kahya 2020). It might also occur when sperm leave the spermatheca, pass through its duct and reach an egg (Harbo 1990). In polyandrous species, sperm competition can favour traits that contribute to fertilisation (Pearcy et al. 2014). Candidate traits include progressive motility, swimming speed, viability, morphology and dimensions such as head, tail and total sperm length (Fitzpatrick and Lüpold 2014; Pearcy et al. 2014). Insect sperm kinematics are still poorly understood. Common measures are curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF) (Lu et al. 2014; WHO 2021). Computer-assisted sperm analysis (CASA) systems typically track the sperm head and are widely used for mammalian sperm (Hook and Fisher 2020; WHO 2021). Insect sperm have much smaller heads and long flagella, which complicate detailed tracking.
After copulation, honey bee sperm mix (Borsuk et al. 2018) and form dense bundles in the spermatheca (Wegener et al. 2014). Over several hours, they can form groups that swim in circles (Tofilski et al. 2018). These changes could reflect interactions or cooperation among spermatozoa, which may contribute to fertilisation in insects (Lüpold et al. 2012; Pearcy et al. 2014). In some insects, sperm cooperation can change motility and swimming speed and may help sperm move through the reproductive tract (Pizzari and Foster 2008; Lüpold et al. 2012; Pearcy et al. 2014; Liberti et al. 2018). Greater motility and speed may come at a cost to viability, which is important during long-term storage.
Sperm movement requires energy and is associated with increased metabolism. Mitochondria produce adenosine triphosphate (ATP) and reactive oxygen species (ROS), which can help regulate movement. If ROS production exceeds antioxidant capacity, oxidative stress can damage sperm structure and function and eventually cause cell death (Reinhardt 2007; Abdelkader et al. 2018). For example, insecticide exposure in adult drones in vivo and in honey bee sperm in vitro increased metabolic activity, as indicated by higher ATP production. Both studies also recorded higher sperm mortality and lower viability (Abdelkader et al. 2015, 2018).
Most in vitro studies of mixing honey bee drone semen found no apparent effect on sperm viability (Shafir et al. 2009; Tofilski et al. 2012; Gençer et al. 2014). Viability also appeared similar in the lateral oviducts and spermatheca during the first four hours, when migration to the spermatheca is greatest in instrumentally inseminated queens (Gençer and Kahya 2011). Work on the consequences of mixing semen from multiple drones during and after mating has largely focused on swimming patterns and viability. Which structural and functional traits are most favourable for fertilisation remains unclear.
In monandrous Hymenoptera, such as most bumble bees and solitary bees, the female mates with one male (Baer 2003). In some species, males form a mating plug that reduces the likelihood of further mating. Sperm from a single male may therefore reach the spermatheca without encountering sperm from other males, making sperm competition in the female reproductive tract less likely (Baer et al. 2006; Strobl et al. 2019).
This preliminary study investigated sperm quality in A. m. capensis drones and how mixing semen from multiple drones might affect sperm behaviour and quality. It compared individual and mixed samples in vitro. The first aim was to determine whether mixing altered motility and kinematic measures. The second was to examine whether structural measures of sperm were associated with the observed function or behaviour.
Materials and methods. See the original article.
Results
This study examined functional and structural sperm traits after mixing drone semen in vitro. Tofilski et al. (2018) previously reported changes in sperm movement over time in queen spermathecae after instrumental and natural insemination. Little forward movement was visible in the first eight hours, although a small fraction of sperm moved rapidly forward. After 16 hours, the researchers observed coordinated circular movement and groups of spermatozoa swimming slowly in circles. The present experiment ended after 60 minutes. Most sperm in the mixed samples moved slowly and non-progressively, and some groups swam in circles. Total motility was initially highest in single- and two-drone samples. It fell markedly in two-drone samples by 60 minutes and declined less in three-drone samples.
Borsuk et al. (2011) similarly found that mixing semen from two mature A. m. carnica drones initially produced very active sperm, but activity subsequently declined. They did not report the interval between observations. In the leaf-cutting ant Acromyrmex echinatior, a male’s own seminal fluid has been reported to produce a smaller increase in sperm motility, possibly helping maintain viability. Mixing it with the seminal fluid of a rival produced a larger increase in motility (Liberti et al. 2018). Total motility comprises progressively (PR) and non-progressively (NP) motile sperm. In two-drone samples, the proportion of non-progressive and slow sperm fell without a corresponding increase in progressive or faster swimmers. Instead, the proportion of immotile sperm rose by more than 50% relative to baseline as total motility declined. Mixing semen from two related drones may therefore have impaired sperm quality in this experiment. Damage to the sperm membrane could affect motility by disrupting the cell’s ability to retain substances needed for movement, such as ATP. A decline in ATP could leave sperm less motile or immotile.
Superoxide dismutase (SOD) acts as an antioxidant and contributes to sperm survival and motility. Therefore, if excess ROS overwhelm the antioxidant defences in semen, oxidative stress can impair sperm function (Reinhardt 2007; Abdelkader et al. 2014, 2018). Oxidative stress may therefore have affected sperm in the two-drone samples. Because viability was not measured over time, the study cannot establish whether the increase in immotile sperm also meant that more sperm had died. Using sperm obtained from seminal vesicles, den Boer et al. (2010) found across six insect species, including honey bees and the leaf-cutting ants Atta colombica and Acromyrmex echinatior, that sperm survival improved with exposure to a male’s own seminal fluid. Survival fell when sperm were exposed to seminal fluid from a related or unrelated male of the same species.The authors suggested that seminal fluid may contain substances with opposing effects, some that impair rival sperm and others that support sperm survival. When Shafir et al. (2009) mixed semen from two unrelated drones and stored it for four days at 25 °C, the mixtures showed slightly higher viability than individual samples, but the difference was not statistically significant. Tofilski et al. (2012) found no difference in sperm viability when they assessed it after collection.
Other studies found substantially lower viability in four-drone semen mixtures (below 50%) than in individual samples (Collins 2004). However, in mixtures of semen from five to eight drones (Collins 2003) or eight to ten drones (Gençer et al. 2014), viability did not change. Tofilski et al. (2012) also found more particles in two-drone samples and proposed a possible role for sperm competition, in which seminal fluid from one drone might cause coagulation and immobilise sperm from another. No coagulation was observed in the present experiment. Differences among studies may reflect collection, storage and sample preparation methods, which can damage sperm (Collins 2004; Shafir et al. 2009; Gençer et al. 2014).
Sperm viability can also vary among individual drones (Collins 2004). Many other factors have been associated with reduced viability, including drone age. Environmental factors such as season, the availability of floral resources and exposure to parasites or pesticides may have a considerable influence (Rangel and Fisher 2019; Yániz et al. 2020; Morais et al. 2022).Varroa infestation levels and the acaricides used to control the mite should also be considered as possible influences on drone sperm viability (Collins 2004; Rinderer et al. 1999). Varroa infestation levels were not measured in the present study. Higher viability in the spermatheca than in ejaculates suggests that viability may play a role in which sperm are stored (Lodesani et al. 2004).
Sperm viability matters in both the drone and the queen, but the mechanisms that preserve it appear to differ. For example, the levels of proteins involved in metabolism, including enzymes, differ considerably between spermathecal and seminal fluid. Spermathecal fluid contains more substances that support sperm survival (Collins et al. 2006; Baer et al. 2009). Dense packing of sperm in the spermatheca may help maintain viability by limiting dilution and, in turn, reducing osmotic stress and endogenous respiration. This can reduce the metabolic activity of densely packed sperm (Reinhardt 2007). In the spermatheca, limiting sperm motility may help preserve viability. Sodium and potassium concentrations are much higher in spermathecal fluid than in drone semen, which may contribute to reduced motility (Verma 1973). Immotile sperm appear more common in older queens, when sperm counts and hypertonicity are lower (Al-Lawati et al. 2009). However, reduced viability in pooled drone semen could be particularly relevant to instrumental insemination because sperm viability may decline faster in instrumentally inseminated queens than in naturally mated queens (Tarpy and Olivarez 2014; Lodesani et al. 2004). Loss of viable sperm can impair reproduction and eventually leave a queen laying unfertilised eggs that develop into drones. Nevertheless, one study found that instrumental insemination with semen of 42.6% viability still allowed queens to lay sufficient fertilised eggs under the conditions tested (Collins 2000).
In contrast to the changes in the two-drone samples, mixing semen from three drones appeared to improve sperm movement over time. In this group, the proportions of non-progressively motile and slow-swimming sperm fell, while the proportions of progressively motile and faster-swimming sperm increased. Most of the resulting values were higher than those in single- and two-drone samples. This was also the only group in which all measured kinematic values increased over time, although the increases were not statistically significant. Increases were most apparent for VSL, LIN and STR. LIN is calculated from VSL and VCL, and STR from VSL and VAP. Higher total motility in honey bee sperm has been associated with greater progressive and rapid motility and higher VCL, VSL, VAP, LIN and STR in individual drone samples from seminal vesicles and ejaculates (Inouri-Iskounen et al. 2020; Murray et al. 2022). The authors propose that concentration, possible sperm cooperation and elapsed time together contributed to the changes observed in three-drone samples. These samples had higher sperm concentrations, which correlated positively with functional measures.
Higher sperm concentration might increase viscosity and affect movement, as suggested by observations in the spermatheca (Tofilski et al. 2018). Al-Lawati et al. (2009) proposed that hypertonicity in the spermatheca might contribute to circular swimming. The authors speculate that hypertonicity could also have been higher in the more concentrated three-drone samples. Experiments with a humpbacked fly and a desert ant found greater sperm swimming speeds in more viscous media (Curtis and Benner 1991; Pearcy et al. 2014). Higher sperm concentration may also favour interactions among spermatozoa. In the eusocial desert ant Cataglyphis savignyi, dozens of spermatozoa form bundles that swim 50% faster than individual sperm. Such cooperation might increase a male’s contribution to the sperm that reach and remain in the spermatheca (Pearcy et al. 2014).
Changes in drone sperm movement have also been attributed to the time between observations, which can allow sperm to settle into stable movement patterns (Tofilski et al. 2018; Yániz et al. 2019). Conditions in a queen’s reproductive tract and spermatheca differ from those in ejaculates.After successful mating or insemination, only a fraction of spermatozoa are stored. Woyciechowski and Król (1996) found that the number stored did not vary with the number of drones whose semen was used for instrumental insemination. In A. m. capensis queens, spermathecal counts appear to range from 3 to 11 million spermatozoa in a volume of about 1 µL. Sample concentrations in all groups fell within this range, but how such variation affects movement in the spermatheca is unknown. Variation in sperm concentration agrees with previous findings in the same subspecies (Murray et al. 2022). A. m. capensis sperm appear shorter than those of other subspecies, such as A. m. carnica, which may affect movement (Gontarz et al. 2016). Sperm in the spermatheca also encounter spermathecal fluid and rely more on anaerobic metabolism than sperm in ejaculates (Liu et al. 2020). Sperm length can influence function and motility in insects (Fitzpatrick and Lüpold 2014). In Drosophila, for example, longer sperm swim more slowly than shorter sperm but can better resist expulsion from the female reproductive tract (Lüpold et al. 2012).
In this experiment, sperm dimensions did not differ among the three groups and were not correlated with motility measures. In the wasp Dahlbominus fuscipennis, five sperm types occur in male seminal vesicles while spermathecae are uniform in size (Lee and Wilkes 1965). Seasonal variation in honey bee sperm dimensions has been reported (Gontarz et al. 2016), but sperm polymorphism among cells that reach and remain in the honey bee spermatheca after mating has yet to be described. The shorter sperm of A. m. capensis may move differently from the longer sperm of other subspecies. A limitation is that drone body size was not measured. Earlier studies associated body size with sperm concentration or the number of spermatozoa per ejaculate (Schlüns et al. 2003; Bratu et al. 2022) and with the occurrence of abnormal sperm (Bratu et al. 2022). The authors’ earlier laboratory work, however, did not suggest an adverse effect of body weight on sperm concentration.
Conclusions
Differences in motility and kinematic measures may reflect a combination of semen mixing, sperm concentration and changes in sperm arrangement over time. The study did not establish a genetic cause. In vitro mixing also excludes the queen’s potential influence on sperm use after mating, including cryptic female choice. In natural mating, only a fraction of the sperm received is stored in the spermatheca. Alongside environmental influences, drone sperm quality may affect queen health and reproduction through post-mating changes, with consequences for colony health and performance (Brutscher et al. 2019).
The findings may be relevant to instrumental insemination programmes and beekeepers. Measures such as concentration, motility and viability can help assess the quality of a pooled semen sample before insemination. Their value as indicators of subsequent queen and colony health warrants further investigation. To clarify the mechanisms behind changes after mixing, the authors propose testing several sperm concentrations, measuring reactive oxygen species and mitochondrial function, and examining sperm traits in the queen’s spermatheca. Routine use in beekeeping would also require simple, affordable methods for assessing drone semen.
Authors
Retha CM Kotzé (Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa)
Gerhard van der Horst (Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa)
Janice F Murray (Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa)
Anja Duminy (Department of Medical Bioscience, University of the Western Cape, Bellville, South Africa, and Applied Research Centre, Langara College, Vancouver, Canada)
Mike Allsopp (Honey Bee Research Section, ARC-Plant Protection Research Institute, Stellenbosch, South Africa)
African Entomology, 2024 · Adapted from the cited article under CC BY 4.0 https://doi.org/10.17159/2254-8854/2024/a17135
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